We propose to examine the control mechanisms regulating early gene expression in Dictyostelium discoideum. Specifically, we will examine the regulation of genes which are induced at or shortly after the onset of development and whose expression is repressed by the start of multicellular differentiation. Three classes of developmentally regulated genes will be examined. The common theme is that the control of their expression is mediated at least in part by cAMP. The first class of genes is induced immediately upon the onset of development and are then repressed by the aggregation stage. In cell culture system, these genes can be precociously repressed by pulsatile signals of cAMP (pulse-repressed genes). The second class of genes is induced several hours after the onset of development and are maximally expressed during aggregation. The expression of these genes is induced by pulsatile signals of cAMP (pulse-induced genes) and are repressed by the addition of continuous levels of cAMP. Inhibitor studies indicate that the regulation of both of these two classes of genes by cAMP is mediated via the cAMP receptor. The third set of genes are the multigene family encoding discoidin I. These genes are induced shortly after the onset of development. Expression of these genes are repressed later toward the end of aggregation by high continuous levels of cAMP. Our aim is to understand the molecular mechanisms controlling these genes. This will be approached by identifying the cis-acting regulatory regions controlling their expression and the identification and purification of trans-acting regulatory proteins. In vitro footprint analysis will be used to identify the cis-acting regions with which putative trans-acting regulatory proteins interact and control the expression of these genes. In addition, a number of molecular approaches will be utilized to identify the genes of these trans-acting factors and for other regulatory genes within the pathways controlling the expression of these genes. The overall goal is to understand the regulatory pathways by which cAMP controls early gene expression either mediated via the cell-surface cAMP receptor or by other molecular mechanisms.